Volume 37, Issue 5, Pages 714-727 (March 2010) Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells  Tomoo Ogi, Siripan Limsirichaikul, René M. Overmeer, Marcel Volker, Katsuya Takenaka, Ross Cloney, Yuka Nakazawa, Atsuko Niimi, Yoshio Miki, Nicolaas G. Jaspers, Leon H.F. Mullenders, Shunichi Yamashita, Maria I. Fousteri, Alan R. Lehmann  Molecular Cell  Volume 37, Issue 5, Pages 714-727 (March 2010) DOI: 10.1016/j.molcel.2010.02.009 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 37, 714-727DOI: (10.1016/j.molcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 UVC Irradiation-Dependent PCNA Ubiquitination in Quiescent Cells and Interaction of DNA Polymerases with NER Postincision Machinery (A–C) Western blot showing ubiquitination of PCNA at indicated times after 20 J/m2 global UVC irradiation (A and B) or 10 mM hydroxyurea (HU) treatment (C) in quiescent cells. Normal (48BR), XPA (XP15BR), and XPC (XP21BR) are all quiescent primary fibroblasts (G0). −, without treatment. SV40-transformed MRC5 cells (MRC5V1) were used as a control. In (B), cells were extracted with Triton X-100 before harvesting (Tx). (D–H) Normal (VH25) or XPA (XP25RO) primary fibroblasts that were either serum starved (G0) or close to confluent density (Cycling) were globally UVC irradiated (20 J/m2) and incubated for 1 hr. Repair proteins were then crosslinked to DNA with formaldehyde treatment followed by ChIP with mouse anti-PCNA, PC-10 antibody (D–F), rabbit anti-polκ, K1 antibody (G), or mouse anti-XRCC1, 33-2-5 antibody (H). Coprecipitated proteins were analyzed by western blotting with the antibodies listed in the Supplemental Experimental Procedures. See also Figure S1. Molecular Cell 2010 37, 714-727DOI: (10.1016/j.molcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Polκ Accumulation at Local Damage Is Dependent on Early Steps of NER (A and B) Non-dividing normal 48BR human primary fibroblasts were transfected with either siNTC non-targeting control (A) or POLK targeting siRNA (B), UVC irradiated (40 J/m2) through a polycarbonate micropore filter (5 μm), followed by 30 min incubation with 10 mM hydroxyurea and immunostaining with mouse anti-RPA70 (RPA70-9; green) and rabbit anti-polκ (K1; red) antibodies. Blue, DAPI stain. (C) XP15BR XP-A cells were cocultured with normal 48BR cells containing blue beads (Ctr), UVC irradiated and processed as in (A), except that the post-UVC incubation was for 1 hr without hydroxyurea. The insets in this and subsequent figures show enlarged images of individual cells. The inset in the white box is a normal cell and in the red box an XP cell. See also Figure S2. Molecular Cell 2010 37, 714-727DOI: (10.1016/j.molcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Role of Polκ in NER Is Dependent on PCNA Ubiquitination (A) siRNA knockdown of RAD18 diminishes the ALD of polκ but not polδ. Cells incubated with blue beads and siRNA non-targeting control were cocultivated with cells incubated with RAD18 siRNA and locally UVC irradiated (5 μm pores, 40 J/m2), followed by 1 hr incubation without inhibitors. White arrows indicate polδ (p125; green) and polκ (red) double positive nuclei in cells treated with non-targeting control (also in inset in white box), whereas green arrows indicate nuclei with polδ spots only in cells treated with RAD18 siRNA (also shown in inset in red box). (B) Western blot showing that siRNA knockdown of RAD18 abolishes PCNA ubiquitination. Normal 48BR primary fibroblasts (left) or normal but SV40 immortalized MRC5VI cells (right) were transfected with either siNTC non-targeting control (NTC) or RAD18 targeting (R18) siRNA and cultured at close to confluent density. Cells were globally UVC irradiated (10 J/m2), followed by incubation for 1 hr without inhibitors. RAD18 and the ubiquitinated PCNA were respectively detected by rabbit anti-RAD18 (Abcam) and mouse anti-PCNA (PC-10) antibodies. Asterisks indicate non-specific bands. (C) ALD of indicated NER proteins in 48BR cells depleted of RAD18 or polκ using siRNAs. Cells were locally UVC irradiated as in (A). Percentage of ALD represents the relative percentage of cells showing ALD of the indicated protein above a predetermined threshold compared with the percentage in relevant controls. Bars and error bars indicate, respectively, averages and standard deviations calculated from at least three independent experiments. (D) ALD of wild-type or indicated mutants of polκ. SV40-transformed MRC5 cells were transfected with plasmid expressing either GFP-tagged wild-type human POLK or POLK with UBZ mutations at the indicated amino acid positions (GFP-POLK; green; see also Figure S2K). Cells were locally UVC irradiated as in (A), followed by immunostaining with anti-RPA antibody (RPA70-9; red). (E) Effect of RAD18 and polκ depletion on UDS. 48BR cells were transfected with indicated siRNAs and UVC irradiated (10 J/m2) followed by EdU incorporation for 2 hr. Bars and error bars, respectively, indicate averages and standard deviations of nuclear fluorescent intensity measured in at least 250 nuclei from at least five different positions. See also Figure S3. Molecular Cell 2010 37, 714-727DOI: (10.1016/j.molcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Effects of DNA Polymerase Knockdowns on UDS and ALD (A) ALD of polδ p125 (A-9 antibody; top and bottom, green), p66 (3E2 antibody; middle, green), and polκ (K1 antibody; bottom, red) in primary 48BR cells treated with the indicated siRNA and locally UVC irradiated (40 J/m2) followed by 30 min incubation. In the top and middle panels, white arrows represent nuclei with spots of indicated polδ subunit, whereas in the bottom panel, green and red arrows indicate nuclei with polδ p125 and polκ spots, respectively. Note that smaller sized green spots seen in the bottom panels are nonspecific nucleoli staining. (B) Histogram analyses are shown. Bars and error bars indicate, respectively, averages and standard deviations of the percentages of ALD calculated from at least three independent experiments shown in (A). (C–E) Effects of multiple DNA polymerase knockdowns on UDS. 48BR cells were transfected with indicated siRNAs and UVC irradiated (10 J/m2) followed by EdU incorporation for 2 hr. Bars and error bars, respectively, indicate averages and standard deviations of nuclear fluorescent intensity measured in at least 250 nuclei from at least five different positions. Molecular Cell 2010 37, 714-727DOI: (10.1016/j.molcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Differential Requirement of Repair Replication Factors for Recruitment of Gap-Filling DNA Polymerases (A) ALD of polδ p125, p66, and polκ in 48BR cells depleted of indicated genes using siRNAs and local UVC irradiation (40 J/m2) followed by 1 hr incubation without inhibitors. Bars and error bars indicate, respectively, averages and standard deviations of the percentages of ALD calculated from at least three independent experiments. (B) Depletion of RFC1 abolishes polδ ALD but does not affect polκ ALD. Cells with non-targeted siRNA cultured with blue beads were cocultured with cells in which RFC1 was depleted by siRNA. White arrow indicates polδ (p125; green) and polκ (red) double positive nuclei, whereas red arrows indicate nuclei with polκ spots only. KD, knockdown (also inset with red box); Ctr, control (also inset in white box). (C) Depletion of XRCC1 abolishes polκ ALD but does not affect polδ ALD. White arrows indicate polδ (p125; green) and polκ (red) double positive nuclei, whereas green arrows indicate nuclei with polδ spots only. (D) UDS following depletion of indicated genes. 48BR cells were transfected with indicated siRNAs and UVC irradiated (10 J/m2) followed by EdU incorporation for 2 hr. Bars and error bars, respectively, indicate averages and standard deviations of nuclear fluorescent intensity measured in at least 250 nuclei from at least five different positions. (E) ALD of polɛ in 48BR cells depleted of indicated genes using siRNAs. Cells were pre-fixed before immunostaining, as described in Figure S4. Bars and error bars indicate, respectively, averages and standard deviations of the percentages of ALD calculated from at least three independent experiments. (F) Depletion of CTF18 inhibits polɛ ALD. Cells with non-targeted siRNA cultured with blue beads were cocultured with cells in which CTF18 was depleted by siRNA. As described in Figure S4, cells were pre-fixed before immunostaining. White arrow indicates polɛ (green) and XPB (red) double positive nuclei, whereas red arrows indicate nuclei with XPB spots only. Neg, polɛ negative non-cycling cells. Molecular Cell 2010 37, 714-727DOI: (10.1016/j.molcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Model for Action of Polκ and Polδ during NER Gap-Filling DNA Synthesis (A) Section of chromatin in quiescent cells with PCNA loaded on the DNA and damage sensed by RAD18, which is then able to ubiquitinate PCNA. (B) Assembly of the preincision complex. (Ca–Ea) In mode 1, following dual incisions to release the damaged fragment (Ca), polɛ is recruited by CTF18-RFC to fill the gap (Da), followed by Ligase I recruitment to seal the nick (Ea). (Cb–Eb) In mode 2, 5′ incision is followed by recruitment of both polδ core by RFC and p66 (Cb) and polκ/XRCC1 by ubiquitinated PCNA (D2). (Eb) After completion of repair synthesis, polκ is released, XPG cleaves off the flap, and XRCC1 recruits Ligase III to seal the remaining nick. Molecular Cell 2010 37, 714-727DOI: (10.1016/j.molcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions